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3296-2ML | LIGHT DIAGNOSTICS SimulFluor® Respiratory Screen Trial, ~50 tests

2 mL  
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      Key ApplicationsDetection Methods
      ICC Fluorescent
      Catalogue Number3296-2ML
      Brand Family Chemicon®
      Trade Name
      • SimulFluor
      • Chemicon
      DescriptionLIGHT DIAGNOSTICS SimulFluor® Respiratory Screen Trial, ~50 tests
      OverviewIntended Use:

      The Light Diagnostics SimulFluor® Respiratory Screen is intended for the screening of the presence of influenza A, influenza B, adenovirus, parainfluenza 1, parainfluenza 2, and parainfluenza 3, and the detection and identification of respiratory syncytial virus (RSV) in respiratory specimens from patients with febrile respiratory illness following specimen amplification in cell culture.

      For In Vitro Diagnostic Use.

      Test Principle:

      Light Diagnostics SimulFluor® Respiratory Screen utilizes direct immunofluorescence to detect and identify RSV in patient specimens, and to screen for the presence of influenza A, influenza B, adenovirus, parainfluenza 1, 2 and 3 viruses in infected cell cultures. The SimulFluor® Respiratory Screen contains two components the primary component is directed against adenovirus, influenza A, influenza B and parainfluenza 1, 2 and 3, while the secondary component is specifically directed against RSV. The monoclonal antibodies in the components will bind to the appropriate viral antigen on the specimen slide. Unbound antibody is washed from the slide with phosphate buffered saline (PBS). The primary component will stain adenovirus, influenza A, influenza B and parainfluenza 1, 2 and 3 infected cells bright apple-green and will not differentiate between these viruses. The secondary component, directed against RSV, will stain RSV-infected cells yellow-gold, thus allowing for the differentiation of RSV from other respiratory viruses in one well. Uninfected cells stain a dull red due to the presence of Evans blue counterstain in the reagent. The SimulFluor® Respiratory Screen is used to discriminate RSV from other respiratory viruses.

      Summary and Explanation:

      Respiratory viruses are responsible for a significant proportion of illness in human populations. Some viruses (e.g. influenza) are seasonal while others (e.g. respiratory syncytial virus (RSV), adenovirus) predominantly affect different age groups. Antiviral therapy is available for some and not all respiratory viruses; rapid screening to identify these organisms allows for early institution of therapy and minimizes the inappropriate use of antibiotics. The two most widely used antiviral agents available for respiratory viruses are amantadine/rimantadine for influenza A, and ribavirin for RSV bronchiolitis. Both agents have differing modes of action, although most affect viral penetration or uncoating (1, 2). At physiological concentrations, amantadine and rimantadine specifically inhibit replication of influenza A (3). Ribavirin has broad spectrum activity in vitro against a host of viruses such as RSV, measles, influenza A and B, and parainfluenza(4, 5).

      Human adenoviruses belong to the family Adenoviridae, genus Mastadenovirus. They are icosahedral, non-enveloped, double stranded DNA viruses 70-90 nm in diameter (6). They have a protein coat of 240 hexon and 12 penton capsomeres. Adenoviruses are responsible for a significant number of clinical respiratory illnesses, particularly in children.

      Upper respiratory diseases in infants and young children can include colds, pharyngitis, and tonsillitis, while lower respiratory illnesses include bronchitis and bronchiolitis (6), and approximately 10% of childhood pneumonia (7). In children under 5, adenovirus is responsible for about 5% of cases of acute respiratory disease (ARD), which is manifested by nasal congestion, coryza, cough and, at times, tonsillitis, fever, and myalgia. The appearance of conjunctivitis with ARD constitutes pharyngoconjunctival fever.

      In addition to respiratory infections, adenoviruses can be a cause of other significant illnesses. Ocular disease resulting from adenovirus infection include epidemic keratoconjunctivitis (EKC), acute hemorrhagic conjunctivitis, and acute follicular conjunctivitis. In addition, adenovirus is related to several gastrointestinal disorders and is probably evident in 7-17% of all childhood gastroenteritis. Types 40 and 41 have been associated with diarrhea and acute gastroenteritis (8). Adenovirus has also been linked with intussusception (9), acute hemorrhagic cystitis (10), and meningoencephalitis.

      Research indicates that the incidence of adenovirus infection in immuno-compromised patients is probably no higher than in normal individuals; however, the severity and probability of death may be greater (11).

      With the exception of types 40 and 41, which grow only in Graham 293 cells, adenovirus can be cultured and isolated in a variety of cell lines such as Hep-2, A549, KB, and HEK. Infection is usually confirmed by immunofluorescence or enzyme immunoassay (EIA). Typical adenovirus cytopathic effects (CPE) manifest as grape-like clusters of rounded, refractive cells with intranuclear inclusions, which appear in 3 to 10 days post-infection(12).

      Influenza viruses are pleomorphic, enveloped viruses, 80-120nm in diameter containing a segmented, single-stranded, negative-sense RNA genome, and are members of the family Orthomyxoviridae (13). Infections with influenza viruses cause highly contagious respiratory disease, which typically results in epidemics (27). There are three types, A, B, and C, in which specificity is conferred by internal nucleoprotein and matrix protein antigens (14).

      Adult infection with influenza viruses may display manifestations ranging from no symptoms to fatal pneumonia (13). More characteristic infections result in tracheobronchitis and small airway involvement (1), with rhinitis and/or pharyngitis. The same spectrum of clinical response can be seen in children with some distinct differences.

      In children, influenza infections usually cause higher fevers, which may be accompanied by febrile convulsions (15-17). Influenza viruses are responsible for 14% of childhood fevers with respiratory tract symptoms severe enough to warrant a physician's attention (15, 17). In addition, children are more likely to experience more gastrointestinal involvement than adults (18) and they develop myositis, otitis media, and croup more frequently (19). Neonatal infection may result in unexplained fever (20) and is potentially fatal (20, 21).

      Lower respiratory tract disease in children and adults associated with influenza infection is manifested in three forms of pneumonia: primary viral pneumonia, combined viral-bacterial pneumonia, and influenza infection followed by bacterial pneumonia (22). Non-pulmonary clinical responses to influenza infection include viremia, cardiac and CNS involvement, Reyes syndrome, and toxic shock.

      Influenza types A and B cause essentially the same spectrum of disease. However, type A infection results in hospitalization approximately four times more often than type B (23), and type B more commonly results in myositis and gastrointestinal involvement (24, 25). Influenza type C rarely results in lower respiratory tract illness but causes sporadic upper respiratory tract disease (23, 26). By adulthood, almost all individuals have antibody to type C (27).

      Detection of influenza A or B from specimens following amplification in culture provides an easy, sensitive and relatively rapid technique for identifying influenza infections. The addition of trypsin to cells makes it possible to culture Influenza viruses in a variety of cell lines such as Madin Darby canine kidney (MDCK), A549 lung carcinoma and primary monkey kidney (PMK) (28, 29). Influenza virus can be detected in culture by immunofluorescence, hemadsorption or hemagglutination techniques using chicken or guinea pig erythrocytes. Cytopathic effect is evident in 3-7 days post-inoculation as vacuolation and cell degradation.

      Parainfluenza viruses, combined with RSV, represent the most significant upper respiratory pathogens in infants and young children (30). Parainfluenza viruses belong to the genus Paramyxovirus of the family Paramyxoviridae. They are enveloped viruses with a single-strand RNA genome of negative polarity and range in diameter from 150-200nm (31). Four types of parainfluenza virus have been identified.

      Parainfluenza types 1 and 2 are major causes of laryngotracheobronchitis (croup). The severity of illness is greatest in children aged 2-4 years (32). Parainfluenza type 3 infection can lead to croup but, most notably, type 3 is second only to RSV as a cause of infant bronchiolitis and pneumonia (33, 34). Illness from type 3 infection is most severe in infants less than 1 year old.

      Severe croup in early childhood or infancy may result in bronchial hyperactivity in older children or adolescents after exercise. However, it remains undetermined whether bronchial hyperactivity was a preexisting condition, which played a role in the pathogenesis of croup or whether it develops as a complication of severe croup (25). In older children and adults, illness may be asymptomatic or mimic the common cold (36).

      Parainfluenza viruses grow well in primary monkey (RMK) or human kidney cell lines and in LLC-MK2, a rhesus kidney heteroploid cell line (38). Trypsin is needed in the medium for the recovery of types 1 and 2 but not type 3. Virus infection of tissue culture can be recognized by hemadsorption of guinea pig erythrocytes. Types 2 and 3 can be recognized by syncytium formation.

      Respiratory Syncytial Virus (RSV) belongs to the family Paramyxoviridae and the genus Pneumovirus. It is an enveloped pleomorphic virus ranging from 150-300nm in diameter (37, 38) with a single-stranded RNA genome (39). By age 2, most children have experienced RSV infections, making it the most important viral cause of childhood lower respiratory tract illness.

      RSV infection usually results in colds with profuse rhinorrhea, but in first-time infections among infants 6 weeks to 6 months old, 25-40% will develop lower respiratory tract illness (37). RSV was responsible for more pneumonia and bronchiolitis than all other microbial pathogens (40). Studies suggest that childhood RSV pneumonia and bronchiolitis may result in long term respiratory abnormalities such as abnormal pulmonary function, asthma, and recurrent cough and bronchitis (41).

      RSV may be detected in patient specimens by immunofluorescence, EIA, or by culture isolation and confirmation (41, 43). A variety of cell lines are suitable for RSV cultivation. For primary isolation, Hep-2 (40) is the most commonly used cell line although others such as Vero, LLC-MK-2, MRC-5 and CV-l have been used. The virus produces characteristic cytopathic effects of syncytium formation and cell destruction.

      Light Diagnostics SimulFluor® Respiratory Screen Kit utilizes monoclonal antibodies to detect and identify RSV in patient specimens following amplification in cell culture, and to screen for the presence of influenza A, influenza B and adenovirus, parainfluenza 1, 2 and 3 following amplification in culture.
      Materials Required but Not Delivered1. Cell culture for isolation of respiratory viruses: Each laboratory must maintain viable stocks of cells at appropriate passage-state that will efficiently allow replication of respiratory viruses from processed patient specimens. These cells must be checked periodically for ability to support growth of respiratory viruses. Appropriate cell lines can be obtained from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852.

      2. Viral transport medium (VTM), which is non-inhibitory to the respiratory viruses and the tissue culture cells used for viral isolation: Hank's balanced salt solution (HBSS) with antibiotics and a protein stabilizer is a suitable medium. Avoid use of animal sera (except precolostral fetal bovine serum (FBS)) as protein stabilizer to prevent interference from inherent antibody.

      3. Tissue culture media such as RPMI or Eagle's Minimum Essential Medium (EMEM) with appropriate amount of precolostral FBS can be used.

      4. Sterile tissue culture tubes, dram vials, or multi-well plates

      5. Acetone, reagent grade or better

      Note: Acetone is hygroscopic and should be kept in tightly stoppered bottles. Presence of moisture in the acetone may result in a hazy appearance on the substrate during fluorescence microscopy.

      6. Acetone-cleaned glass slides with wells at least 6mm in diameter in a hydrophobic mask

      7. Sterile pipettes

      8. Humid chamber

      9. Sodium hypochlorite solution (0.05%)

      10. No. 1 coverslips

      11. 37°C incubator with rheostat for temperature regulation

      12. Sterile swabs

      13. Forceps

      14. Vials for collection and transportation of specimens

      15. Fluorescence microscope with appropriate filter combination for fluorescein (excitation peak 490 nm, emission peak 520 nm)

      16. Sterile glass beads (1-3 mm diameter)

      17. Centrifuge

      18. Vortex mixer or sonicator

      19. Distilled or deionized water
      Product Information
      • SimulFluor® Respiratory Screen - (Catalog No. 5296-2 mL). One 2 mL dropper vial containing a primary component specific for adenovirus, influenza A, influenza B, parainfluenza 1, 2 and 3, and a secondary component specific for RSV, protein stabilizer, Evans blue and 0.1% sodium azide (preservative).
      • Respiratory Control Slides - (Catalog No. 5071). Two Control Slides containing seven positive wells each infected with a different respiratory virus. (Adenovirus, influenza A, influenza B, parainfluenza 1, parainfluenza 2, parainfluenza 3 and respiratory syncytial virus) and one uninfected well.
      • PBS - (Catalog No. 5087). One packet of Phosphate-Buffered Saline (PBS) yields 1 liter when dissolved in distilled water. Store in a clean closed container at room temperature.
      • Tween 20 / Sodium Azide Solution (100X) - (Catalog No. 5037). One 10 mL vial containing (Tween 20) and sodium azide concentrate to be diluted 1:100 in PBS.
      • Mounting Fluid - (Catalog No. 5013). One 10 mL dropper vial containing buffered glycerin at pH8.5, fluorescence enhancer, and sodium azide as preservative. Store at room temperature.
      Detection methodFluorescent
      Key Applications
      • Immunocytochemistry
      Biological Information
      Physicochemical Information
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • For in vitro Diagnostic Use
      • CE Mark
      Storage and Shipping Information
      Storage ConditionsWhen stored at 2-8°C, the SimulFluor® Respiratory Screen is stable up to the expiration date printed on the kit label. Do not freeze or expose to elevated temperatures. Discard any remaining reagent after the kit expiration date.

      Warnings and Precautions:

      · Sodium azide (present in the conjugate, monoclonal antibodies, wash buffer, and mounting fluid) can react with lead or copper plumbing to form potentially explosive metal azides. When disposing of these materials, flush with large volumes of water to prevent azide build-up.

      · Pooling or diluting conjugates or monoclonal antibodies may cause erroneous results.

      · Do not allow slides to dry at any time during the staining procedure.

      · Handle all specimens and materials as if potentially infectious. Decontaminate with 0.05% sodium hypochlorite (a 1:100 dilution of household bleach) prior to disposal.

      · Do not expose reagents to bright light during storage or incubation.

      · Avoid contact with Evans blue (present in the SimulFluor® Respiratory Screen) as it is a potential carcinogen. If skin contact occurs, flush with large volumes of water.

      · Acetone is extremely flammable and harmful if swallowed or inhaled. Keep away from heat, sparks, or flames. Avoid breathing vapor. Use adequate ventilation.

      · Do not mouth pipette reagents.

      · Do not substitute reagents from other manufacturers.

      · Alteration of protocol provided may cause erroneous results.

      · When staining multiple samples on a slide, avoid cross contamination between samples.

      · Mounting Fluid contains a fluorescence enhancer that may be destructive to mucous membranes. Avoid direct skin or mucous membrane contact. If contact occurs, flush with large volumes of water.
      Packaging Information
      Material Size2 mL
      Transport Information
      Supplemental Information